TOR-autophagy signaling in adult zebrafish models of cardiomyopathy

The target of rapamycin (TOR) kinase is part of an evolutionarily conserved signaling pathway that coordinates cell growth, survival, and autophagy. Previously, pharmacological studies using rapamycin have suggested a cardioprotective effect of TOR signaling inhibition on cardiomyopathy. We found that rapamycin exerts a conserved cardioprotective effect in two adult zebrafish models of cardiomyopathy of different etiology, and provided the first genetic evidence to support a long-term cardioprotective effect of TOR signaling inhibition. Moreover, we detected dynamic TOR-autophagy activities along different stages of cardiomyopathy. This needs to be considered when developing TOR-autophagy-based therapeutics for cardiomyopathy.     

Inhibition of protein synthesis by TOR inactivation revealed a conserved regulatory mechanism of the BiP chaperone in Chlamydomonas

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.     

Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii

The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.     

Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants

Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs.     

Plant target of rapamycin signaling network: Complexes,conservations, and specificities

Target of rapamycin (TOR) is an evolutionarily conserved protein kinase that functions as a central signaling hub to integrate diverse internal and external cues to precisely orchestrate cellular and organismal physiology. During evolution, TOR both maintains the highly conserved TOR complex compositions, and cellular and molecular functions, but also evolves distinctive roles and strategies to modulate cell growth, proliferation, metabolism, survival, and stress responses in eukaryotes. Here, we review recent discoveries on the plant TOR signaling network. We present an overview of plant TOR complexes, analyze the signaling landscape of the plant TOR signaling network from the upstream signals that regulate plant TOR activation to the downstream effectors involved in various biological processes, and compare their conservation and specificities within different biological contexts. Finally, we summarize the impact of dysregulation of TOR signaling on every stage of plant growth and development, from embryogenesis and seedling growth, to flowering and senescence.     

Two TOR complexes,only one of which is rapamycin sensitive,have distinct roles in cell growth control

The target of rapamycin (TOR) proteins in Saccharomyces cerevisiae, TOR1 and TOR2, redundantly regulate growth in a rapamycin-sensitive manner. TOR2 additionally regulates polarization of the actin cytoskeleton in a rapamycin-insensitive manner. We describe two functionally distinct TOR complexes. TOR Complex 1 (TORC1) contains TOR1 or TOR2, KOG1 (YHR186c), and LST8. TORC2 contains TOR2, AVO1 (YOL078w), AVO2 (YMR068w), AVO3 (YER093c), and LST8. FKBP-rapamycin binds TORC1, and TORC1 disruption mimics rapamycin treatment, suggesting that TORC1 mediates the rapamycin-sensitive, TOR-shared pathway. FKBP-rapamycin fails to bind TORC2, and TORC2 disruption causes an actin defect, suggesting that TORC2 mediates the rapamycin-insensitive, TOR2-unique pathway. Thus, the distinct TOR complexes account for the diversity, specificity, and selective rapamycin inhibition of TOR signaling. TORC1 and possibly TORC2 are conserved from yeast to man.     

The fission yeast TOR homolog, tor1+, is required for the response to starvation and other stresses via a conserved serine

Targets of rapamycin (TORs) are conserved phosphatidylinositol kinase-related kinases that are involved in the coordination between nutritional or mitogenic signals and cell growth. Here we report the initial characterization of two Schizosaccharomyces pombe TOR homologs, tor1(+) and tor2(+). tor2(+) is an essential gene, whereas tor1(+) is required only under starvation and other stress conditions. Specifically, Deltator1 cells fail to enter stationary phase or undergo sexual development and are sensitive to cold, osmotic stress, and oxidative stress. In complex with the prolyl isomerase FKBP12, the drug rapamycin binds a conserved domain in TORs, FRB, thus inhibiting some of the functions of TORs. Mutations at a conserved serine within the FRB domain of Saccharomyces cerevisiae TOR proteins led to rapamycin resistance but did not otherwise affect the functions of the proteins. The S. pombe tor1(+) exhibits different features; substitution of the conserved serine residue, Ser(1834), with arginine compromises its functions and has no effect on the inhibition that rapamycin exerts on sexual development in S. pombe.     

Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation

Background

Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.

Findings

We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH _2-46/ΔCT₁₀₄).

Conclusions

These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.     

TOR signaling in growth and metabolism

The target of rapamycin (TOR) is a conserved Ser/Thr kinase that regulates cell growth and metabolism in response to environmental cues. Here, highlighting contributions from studies in model organisms, we review mammalian TOR complexes and the signaling branches they mediate. TOR is part of two distinct multiprotein complexes, TOR complex 1 (TORC1), which is sensitive to rapamycin, and TORC2, which is not. The physiological consequences of mammalian TORC1 dysregulation suggest that inhibitors of mammalian TOR may be useful in the treatment of cancer, cardiovascular disease, autoimmunity, and metabolic disorders.     

Target of rapamycin (TOR) in nutrient signaling and growth control

TOR (Target Of Rapamycin) is a highly conserved protein kinase that is important in both fundamental and clinical biology. In fundamental biology, TOR is a nutrient-sensitive, central controller of cell growth and aging. In clinical biology, TOR is implicated in many diseases and is the target of the drug rapamycin used in three different therapeutic areas. The yeast Saccharomyces cerevisiae has played a prominent role in both the discovery of TOR and the elucidation of its function. Here we review the TOR signaling network in S. cerevisiae.     

Complexity of the TOR signaling network

The target of rapamycin (TOR) is a serine/threonine kinase of the phosphatidylinositol kinase-related kinase family and is highly conserved from yeast to mammals. TOR functions as a central regulator of cell growth and is itself regulated by a wide range of signals, including growth factors, nutrients and stress conditions. Recent studies in eukaryotic cells have identified two distinct TOR complexes, TORC1 and TORC2, which phosphorylate different substrates and have distinct physiological functions. Here, we discuss new findings that have extended the complexity of TOR signaling and the different roles of the TORC complexes in yeast, flies and mammals.     

Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae

The target of rapamycin (TOR) is serine/threonine protein kinase that is highly conserved among eukaryotes and can be inactivated by the antibiotic rapamycin through the formation of a ternary complex composed of rapamycin and two proteins, TOR and FKBP12. Differing from fungi and animals, plant FKBP12 proteins are unable to form the ternary complex, and thus plant TORs are insensitive to rapamycin. This has led to a poor understanding of TOR functions in plants. As a first step toward the understanding of TOR function in a rapamycin-insensitive unicellular red alga, Cyanidioschyzon merolae, we constructed a rapamycin-susceptible strain in which the Saccharomyces cerevisiae FKBP12 protein (ScFKBP12) was expressed. Treatment with rapamycin resulted in growth inhibition and decreased polysome formation in this strain. Binding of ScFKBP12 with C. merolae TOR in the presence of rapamycin was demonstrated in vivo and in vitro by pull-down experiments. Moreover, in vitro kinase assay showed that inhibition of C. merolae TOR kinase activity was dependent on ScFKBP12 and rapamycin.     

The Opposing Actions of Target of Rapamycin and AMP-Activated Protein Kinase in Cell Growth Control

Cell growth is a highly regulated, plastic process. Its control involves balancing positive regulation of anabolic processes with negative regulation of catabolic processes. Although target of rapamycin (TOR) is a major promoter of growth in response to nutrients and growth factors, AMP-activated protein kinase (AMPK) suppresses anabolic processes in response to energy stress. Both TOR and AMPK are conserved throughout eukaryotic evolution. Here, we review the fundamentally important roles of these two kinases in the regulation of cell growth with particular emphasis on their mutually antagonistic signaling.An efficient homeostatic response to maintain cellular energy despite a noncontinuous supply of nutrients is crucial for the survival of organisms. Cells have, therefore, evolved a host of molecular pathways to sense both intra- and extracellular nutrients and thereby quickly adapt their metabolism to changing conditions. The target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) signaling pathways control growth and metabolism in a complementary manner with TOR promoting anabolic processes under nutrient- and energy-rich conditions, whereas AMPK promotes a catabolic response when cells are low on nutrients and energy. Both pathways are highly conserved from yeast to human. This review summarizes the cross talk between TOR and AMPK in different organisms.     

Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway

Identification of the cellular targets of small-molecule hits in phenotypic screens is a central challenge in the development of small molecules as biological tools and potential therapeutics. To facilitate the process of small-molecule target identification, we developed a global, microarray-based method for monitoring the growth of pools of yeast strains, each overexpressing a different protein, in the presence of small molecules. Specifically, the growth of Saccharomyces cerevisiae strains harboring approximately 3,900 different overexpression plasmids was monitored in the presence of rapamycin, which inhibits the target of rapamycin (TOR) proteins. TOR was successfully identified as a candidate rapamycin target, and many additional gene products were implicated in the TOR signaling pathway. We also characterized the mechanism of LY-83583, a small-molecule suppressor of rapamycin-induced growth inhibition. These data enabled functional links to be drawn between groups of genes implicated in the TOR pathway, identified several candidate targets for LY-83583, and suggested a role for mitochondrial respiration in mediating rapamycin sensitivity.     

Growth control via TOR kinase signaling,an intracellular sensor of amino acid and energy availability,with crosstalk potential to proline metabolism

The TOR (Target of Rapamycin) protein kinase pathway plays a central role in sensing and responding to nutrients, stress, and intracellular energy state. TOR complex 1 (TORC1) is comprised of TOR, Raptor, and Lst8 and its activity is sensitive to inhibition by the macrolide antibiotic rapamycin. TORC1 regulates protein synthesis, ribosome biogenesis, autophagy, and ultimately cell growth through the phosphorylation of S6 K, 4E-BP, and other substrates. As TORC1 activity is positively or negatively modulated in response to upstream regulators, cellular growth rate is, respectively, enhanced or suppressed. A separate multiprotein TOR complex, TORC2, is insensitive to direct inhibition by rapamycin and does not regulate growth patterns directly; TORC2 can, however, impact certain aspects of TORC1 signaling and cell survival. TOR signaling is an ancient pathway, conserved among the yeasts, Dictyostelium, C. elegans, Drosophila, mammals, and Arabidopsis. This review will focus on the regulation of TORC1 in mammalian cells in the context of amino acid sensing/regulation and intracellular ATP homeostasis, but will also include comparisons among other organisms.     

The Target of Rapamycin Signaling Pathway Regulates mRNA Turnover in the Yeast Saccharomyces cerevisiae

The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes. We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae. During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected. Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion. The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway. Of the destabilized mRNAs, two different responses to rapamycin were observed. Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure. Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation. In contrast, the mRNAs that respond slowly are destabilized by rapid decapping. In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms.